For convenient and efficient testing of clinical samples of biological fluids, small precise quantities of stable diagnostic reagents are needed. These reagents must be efficiently and economically prepared in large quantities without sacrificing precise delivery of the reagents. Further, the reagents should be delivered to the user in a stabilized form so as to prevent wastage of expensive reagents. The form in which the reagents are provided must allow simple rapid testing without the intervention of highly skilled technicians. One form which can meet these needs is a tablet containing all of the reagents necessary to conduct a given diagnostic assay.
A tablet of this type or one used for therapeutic purposes needs to be stable, easily prepared in a highly reproducible manner, and to dissolve rapidly upon mixing with an appropriate sample. Many difficulties have been recognized in preparing tablets containing diagnostic or therapeutic reagents. There must be good tablet-to-tablet reproducibility which, in turn, means that the dry powder blend from which the tablets are made must be highly homogeneous, the reagents must withstand the conditions used to prepare the powder blend and then the tablets, and the resulting tablet must be easily dissolved in aqueous solutions.
Preferred tablets for use in diagnostic applications are very small, preferably less than 50 mg and more preferably less than 10 mg. The need for such small tablets compounds the normally difficult problems of producing tablets useful as carriers of diagnostic reagents. The problem of inhomogeneity of the dry powder blends used to form the tablets is particularly severe since even minor inhomogeneities have a large adverse effect on the tablet-to-tablet reproducibility. This is so because of the relatively small number of dry powder particles needed to form each tablet. In order to obtain the necessary homogeneity, techniques such as certain spray freeze processes, are required (see, for example, U.S. Pat. No. 3,932,943, issued Jan. 20, 1976, and U.S. Pat. No. 3,721,725, issued Mar. 20, 1973 both to Briggs et al.).
The proportion of active reagents in a tablet formulation is often substantially smaller than that of the inert materials required to form a compact tablet of reasonable size. Further, the pure active reagents may not possess the attributes needed to insure proper formation of compressed tablets. Tablet ingredients, in general, must have some of the following attributes: flowability, lubricity, anti-adherence, bulking and compactability. Certain non-active materials can be added to a tablet formulation to contribute one or more of these attributes; these are called excipients. Any excipients used in a given application must not interfere with the desired action of the active reagents either physically or chemically. A wide variety of excipients are known and the selection of an appropriate excipient for a given application is a combination of known chemical relationships and trial-and-error experimentation.
Tablets prepared as carriers of diagnostic reagents and some therapeutic tablets must meet several additional criteria unique in compressed tablets. High levels of tablet-to-tablet reproducibility are required to assure accurate results. Frequently, the tablets are used to form aqueous solutions of reagents that react with components of biological fluids to form a colored product or other optically detectable signal. This requires that the excipient chosen be completely water soluble so as to avoid insoluble particles which might block the optical light thereby generating false results. Partial insolubility might also cause problems if not all the active reagents are released into the solution thereby creating tablet-to-tablet inconsistencies. Rapid dissolution is also a frequent requirement so that the desired chemical reaction can take place within a given time period. Thus, the production of a small reagent tablets requires: the ability to produce a uniform, stable, dry reagent powder, and the ability to produce a firm, precise, rapidly and easily dissolved tablet from the reagent powder.
An additional consideration in the selection of a tablet excipient is the need for it to be compatible with the process selected for preparing the dry powder blend to be tableted. The so-called S-1 spray freezing process described by Briggs et al., see above, requires that the spray solution have a relatively high percent solids content. 25-40%, in order to produce a powder of sufficient bulk density to allow for proper tableting. The preferred excipient disclosed by Briggs et al. is mannitol. Mannitol, however, can only achieve the required percent solids content when the solution is warmed to about 30.degree. C. This elevated temperature is frequently detrimental to the active reagents, thus limiting the applicability of this technique.
Another requirement for using the S-1 technique is that the frozen droplets formed must be able to be lyophilized without melt back or formation of syrups or amorphous masses. Mannitol is preferred in this respect relative to other saccharides, such as sucrose, which while highly soluble are very diffcult to lyophilize successfully.
Thus, there is a need for an improved excipient for use in preparation of a dry powder blend by the S-1 spray-freeze process which can be used to form tablets containing diagnostic or therapeutic reagents. This excipient should have the following properties: high solubility in aqueous solutions at or below room temperature; ability to lyophilize the resulting frozen mass without formation of syrups or amorphous masses; rapid redissolution upon addition of water or aqueous biological fluids; and the ability to enhance the stability of the active reagents to be delivered by the tablets.
In addition to active ingredients and excipients, other ingredients such as stabilizers may also be added to a tablet formulation. Stabilizers may perform two types of functions. They may permit lyophilization of certain ingredients which would not ordinarily survive lyophilization and/or they may extend the storage stability of the tablet prepared. Some stabilizers can perform only one of these functions, while others perform both. By storage stability is meant the time period after the tablet has been made during which the tablet performs acceptably. It is expected that essentially all tablets will eventually show degradation of one or more of the active ingredients, thereby limiting the storage stability.
It is known that proteins can be stabilized by lyophilization (Kozlov et al., GB No. 2,049,700B, issued Dec. 31, 1980). It is also known that certain poly-hydroxy compounds, including saccharides are useful in stabilizing asparaginase during lyophilization [Hellman et al., Biochem. Biophys. Acta, Volume 749, 133-142 (1983)]. Lee et al. [J. Biol. Chem., Volume 256(14), 7193-7201 (1981)] also reported that sucrose is useful in stabilizing chymotrypsin, chymotrypsinogen and ribonuclease during lyophilization. These procedures are limited in that bulk lyophilizates are not generally suitable for use in small tablets due to inhomogeneities in the dried powders.
There is a report that trehalose (.alpha.-D-glucopyranosyl-.alpha.-D-glucopyranoside) is effective in stabilizing lipid membrane structures upon lyophilization [e.g., Crowe et al., Science, Volume 223, 701-703 (1984)]. It has also been reported that trehalose is effective in stabilizing Tumor Necrosis Factor (Hayashi et al., U.S. Pat. No. 4,457,916, issued July 3, 1984) and menningococcal polysaccharides (Guthohrlein et al., U.S. Pat. No. 4,206,200 issued June 3, 1980) during lyophilization. These uses of trehalose as a stabilizer, while allowing lyophilization, did not lead to tabletable powders due to the use of bulk lyophilization techniques.
Diagnostic reagents have been stabilized in the past by lyophilization under certain conditions [Rush et al., U.S. Pat. No. 3,819,488, issued June 25, 1974] but, as before, the use of bulk lyophilization limits the usefulness of these methods because the resulting powders do not afford homogeneous tablets.